Treatment for BK polyomavirus infection

ABSTRACT

The present disclosure provides novel compositions and methods for treating infection by a viral pathogen, e.g., a BK or JC polyomavirus, using agents having sialidase activity. In particular, the present disclosure provides methods that entail administering agents having an anchoring domain that anchors the compound to the surface of a target cell, and a sialidase domain that can act extracellularly to inhibit infection of a target cell by a pathogen.

RELATED APPLICATIONS

This application is the U.S. national stage under 35 USC § 371 ofInternational Application Number PCT/US2014/041767, filed on Jun. 10,2014, which claims priority to U.S. Application No. 61/833,394, filed onJun. 10, 2013, the entire contents of which is hereby incorporated byreference.

BACKGROUND

Several types of polyomavirus gain cell entry by recognizing a sialicacid-containing component on the cell surface for initial attachment andsubsequent association with the cells.

BK polyomavirus (BKV or BKPyV) is latent in most individuals of thegeneral population. However, late onset haemorrhagic cystitis, BKVnephropathy, and ureteral stenosis are all associated with BKVreactivation, for example, in immunosuppressed individuals. Lyticinfection resulting from reactivation is most commonly noted in renaland bone marrow transplant patients, respectively (S. D. Gardner et al.(1971) Lancet 1253; L. K. Dropulic et al. (2008) Bone Marrow Transplant.41: 11-18.) Two forms of the virus, archetype virus and rearrangedvariants, exist and are identified based on the DNA sequence structureof the non-coding control region. Archetype virus can be commonlyisolated from the urine of both healthy individuals, as a result oftransient reactivation events, as well as patients with disease (M. J.Imperiale, et al (Eds.). (2007) Fields Virology, Lippincott Williams &Wilkins, pp. 2263-2298; K. Doerries et al. (2006) Adv. Exp. Med. Biol.577: 102-116.). In contrast, rearranged variants are most often found inthe serum of patients with BKPyV-associated diseases.

BKPyV infection can occur through a host of different cell types andtissue throughout the body, however the main site of BKPyV reactivationand replication is within the kidneys and urinary tract. The virus isable to reactivate in renal transplant and bone marrow transplantpatients, furthermore, BKPyV reactivation and disease, albeit rarely,have been observed in other immunocompromised conditions includingsystemic lupus erythematosus noted in other solid organ transplantrecipients, in patients with HIV/AIDS (M. Jiang et al. (2009) Virology384:266-273), fatal pneumonia, native kidney nephritis and encephalitis(A. Galan et al. (2005) Hum. Pathol. 36:1031-1034; E. S. Sandler et al.(1997) Bone Marrow Transplant. 20: 163-165; 0. Cubukcu-Dimopulo et al.(2000) Am. J. Surg. Pathol. 24: 145-149.)

A non-enveloped virus, BKPyV is comprised of essentially a proteincapsid and DNA. It consists mainly of the building blocks that serve asthe structure of the viral capsid: VP1, VP2 and VP3. The capsidsurrounds the double-stranded circular DNA genome integrated withhistones. BKPyV infection is noted in cells with cell surfacegangliosides.

Currently, treatment of polyomavirus-associated nephropathy consists ofreducing immunosuppression in the transplant patients. Additionally,other known drug therapies for infections of DNA viruses have beentested for efficacy towards BKPyV infections including cidovir,leflunomide, and fluoroquinolones (S. Safrin et al. (1997) Rev. Med.Virol. 7:145-156; M. A. Josephson et al. (2006) Transplantation 81:704-710. S. Gabardi, S. S. et al. (2010) Clin. J. Am. Soc. Nephroi. 5:1298-1304.)

JC polyomavirus infection is species specific and only noted in humans.The cellular receptors recognized by JC virus include the N-linkedglycoprotein with an alpha (2,6)-linked sialic acid 12 present on manyhuman cells and the serotoninergic 5HT_(2a) receptor in permissiveastroglial cell cultures (Komagome R et al. (2002) J Virol.76:12992-3000). 5HT_(2a) is present in several tissue, including thekidney, on epithelial cells, in the blood on B lymphocytes andplatelets, and in the CNS on glial cells and neurons (Gray J A et al.(2001) Mol Pharmacol. 60:1020-30; Fonseca M I et al. (2001) Brain ResMol Brain Res. 89:11-9). JCV infection has a narrow host cell range; JCVDNA has been detected in oligodendrocytes, astrocytes, lymphocytes,kidney epithelium cells, tonsil stromal cells, and plasma cells (MonacoM C et al. (1998) J Virol. 72:9918-23; Tan, C S et al. (2009) J InfectDis.). JCV pathogenesis largely occurs in immunocompromised settings andleads to a number of CNS pathologies including progressive multifocalleukoencephalopathy (PML), granule cell neuronopathy (GCN),encephalopathy and meningitis (Tan, C et al. (2011) Lancet Neurol. 9:425-437).

PML is a demyelinating disease of the CNS caused by the reactivation ofthe human JC polyomavirus (JCV or JC PyV). Often fatal, PML is aconsequence of a productive JCV infection of oligodendrocytes and to alesser extent, astrocytes some of which harbor late JCV genes and aredestroyed, while others withstand a failed infection and appeartransformed. Symptoms vary and include weakness, sensory deficit,hemianopsia, cognitive dysfunction, aphasia, or coordination and gaitdifficulties. Alternatively, GCN arises from JC infection and subsequentdestruction of granule cell neurons in the cerebellum (Koralnik I J etal. (2005) Ann Neurol. 57:576-80.). GCN patients present with subacuteor chronic onset of cerebellar dysfunction, including gait ataxia,dysarthria and incoordination, and show cerebellar atrophy upon MRIanalysis.

The JC virus is a polyomavirus which harbors a circular enclosed doublestranded DNA from which the genes responsible for viral transformation,gene regulation, and replication are encoded counterclockwise and thenon-coding regulatory region and the late genes for the agnoprotein andthe viral capsid proteins, are encoded clockwise. The coding regionconsists of 90% of the viral sequence. Following primary infection, JCVcan remain dormant in the kidneys, bone marrow and lymphoid tissue(Monaco M C et al. (1996) J Virol. 70:7004-12; Tan C S et al. (2009) JInfect Dis. 199:881-8; Randhawa P et al. (2005) J Infect Dis.192:504-9).

JCV infection is mainly diagnosed in immunocompromised patients. PML isa fatal disease and although there is no specific treatment,immunocompromised patients i.e., HIV patients, susceptible to JCV arenoted to have a better prognosis with the advent of combinationantiretroviral therapy. Monoclonal antibody therapies for autoimmunedisorders, including natalizumab for multiple sclerosis, efalizumab forpsoriasis and rituximab for non-Hodgkin lymphoma, have also beenassociated with the onset of PML resulting from JCV infection due totheir suppression of the immune system (FDA MedWatch: US Dept of Healthand Human Services. 2009; Schwab N et al. (2009) Multiple Sclerosis15:S271-S7).

SUMMARY

In one aspect, the disclosure provides a method for treating infectionby a pathogen. In preferred embodiments, the method comprisesadministering an agent having sialidase activity, such as a sialidase ora fragment thereof containing a sialidase catalytic domain, including asialidase catalytic domain fusion protein, to a subject to treat aninfection. A pathogen can be a viral pathogen, e.g., a polyomavirus. Thepathogen can bind to a sialic acid-containing component on the surfaceof a target cell. The method includes administering an effective amountof an agent of the present disclosure to at least one target cell of asubject. Preferably, the pharmaceutical composition can be administeredby the use of a topical formulation.

In some cases the agent includes a glycosaminoglycan (GAG) bindingdomain. The GAG binding domain can be all or a fragment of: humanplatelet factor 4, human interleukin 8, human antithrombin III, humanapoprotein E, human angio associated migratory protein, or humanamphiregulin.

The source of the sialidase activity can be bacterial or human. Inpreferred embodiments, the bacterial source of the sialidase is selectedfrom Vibrio cholera, Arthrobacter ureafaciens, Clostridium perfringens,Actinomyces viscosus, and Micromonospora viridifaciens.

In the above method, administration of the agent having sialidaseactivity leads to an improvement in the parameters resulting from theinfection.

In some embodiments, the agent comprises, consists of, or consistsessentially of all or a catalytically active portion of a sialidase.

In some cases, the agent having sialidase activity comprises, consistsof, or consists essentially of DAS181. In some cases, the methodcomprises administering composition comprising microparticles comprisinga compound that comprises, consists of, or consists essentially ofDAS181.

In another aspect, the present disclosure provides new compositions andmethods for treating BK polyomavirus infection and disorders associatedwith BK polyomavirus (BKV or BK PyV) infection. Specifically, itprovides compounds which can act extracellularly to reduce or preventinfection of a cell by a BKV. Some preferred embodiments of thedisclosure include therapeutic compounds having an anchoring domain thatfacilitates association of the compound with the surface of a targetcell and a sialidase domain that can act extracellularly to reduce orprevent infection of the target cell by a pathogen, such as a virus. Insome embodiments the compound comprises, consists of or consistsessentially all or a catalytically active portion of a sialidase.

Described herein are methods of treating an infection by BK polyomavirusor a BK polyomavirus associated disorder in a patient, the methodcomprising administering to the patient a therapeutically effectiveamount of an agent having sialidase activity. In various embodiments:the patient is immunocompromised; the patient has undergonehaematopoietic stem cell transplant or is being prepared forhaematopoietic stem cell transplant; the disorder is BKV nephropathy;the disorder is nephritis; disorder is hemorrhagic cystitis; thedisorder is ureteral stenosis; the patient has undergone solid organtransplant or is being treated in preparation for solid organtransplant; the disorder is lupus; the agent having sialidase activityis a polypeptide comprising a portion of a sialidase having sialidaseactivity. In some cases, the polypeptide comprises or consists of afusion protein wherein the fusion protein comprises at least a firstportion comprising a portion of a sialidase having sialidase activityand a second portion binds to a glycosaminoglycan (GAG). In some cases,the polypeptide comprises or consists of a fusion protein comprising atleast a first portion comprising a portion of a sialidase havingsialidase activity and a second portion has a net positive charge atphysiological pH. In some cases, the portion that binds to a GAG isselected from the group comprising: human platelet factor 4 (SEQ ID NO:2), human interleukin 8 (SEQ ID NO: 3), human antithrombin III (SEQ IDNO: 4), human apoprotein E (SEQ ID NO: 5), human angio associatedmigratory protein (SEQ ID NO: 6), and human amphiregulin (SEQ ID NO: 7).In some cases, the agent having sialidase activity is a bacterialsialidase (e.g., the bacterial sialidase is selected from a groupcomprising: Vibrio cholera, Arthrobacter ureafaciens, Clostridiumperfringens, Actinomyces viscosus, and Micromonospora viridifaciens). Insome cases, the agent having sialidase activity is a human sialidase.

In some cases, the agent is administered to the kidneys; the agent isadministered to the ureter; the agent is administered to the bladder;the agent is administered topically (e.g., to one or more surfaces ofthe bladder, kidney or ureter). In some cases, the agent is administeredby infusion into the kidneys; the agent is administered by infusion intothe bladder; and the agent is administered by catheter into the bladder.

In some cases the administration of the agent having sialidase activitycauses one or more of: a reduction of dysuria, a reduction of frequencyof urination, a reduction of subrapublic pain, a reduction of hematuria,a decrease in symptoms associated with nephropathy, and/or a reductionof BKPyV viral load.

In some cases, the infection is associated with an event selected fromthe group comprising: commencement of immunosuppressive therapy, renaltransplant and bone marrow transplant.

In yet another aspect, the present disclosure provides new compositionsand methods for treating JC virus infection and disorders associatedwith JC polyomavirus (JCV or JC PyV) infection. In some cases, the JCpolyomavirus is actively replicating in the subject. Specifically, itprovides compounds which can act extracellularly to reduce or preventinfection of a cell by a JC virus. Some preferred embodiments of thedisclosure include therapeutic compounds having an anchoring domain thatfacilitates association of the compound with the surface of a targetcell and a sialidase domain that can act extracellularly to reduce orprevent infection of the target cell by a pathogen, such as a virus. Insome embodiments, the compound comprises, consists of, or consistsessentially of all or a catalytically active portion of a sialidase. Insome embodiments, the compound is formulated as a wafer.

In various embodiments: the disorders associated with JC polyomavirus isprogressive multifocal leukoencephalopathy, granule cell neuronopathy,encephalopathy, meningitis, or immune reconstitution inflammatorysyndrome.

In some cases, the agent is administered to the brain or spinal cord. Insome cases, the agent is administered topically (e.g., by injection intothe brain, or cerebrospinal fluid).

In some cases, the administration of the agent having sialidase activitycauses one or more of: an improvement in the myelination in the brain,an improvement in the myelination in the spine, an increase in strength,a reduction of sensory deficit, a reduction of hemianopsia, animprovement in of cognitive function, a reduction of aphasia, areduction of gait ataxia, a reduction of dysarthria, an improvement incoordination, a decrease of PML lesions, and a reduction of JCV viralload.

DETAILED DESCRIPTION

In general, the present disclosure relates to methods for treating viralinfection using agents having sialidase activity. Suitable agents aredescribed in U.S. Pat. Nos. 8,084,036 and 7,807,174 which are bothhereby incorporated by reference in their entirety. The agents havingsialidase activity can remove sialic acid residues from the surface ofcells and reduce in infection by certain viruses that binding to sialicacid residues, e.g., BK polyomavirus (BKV, BK PyV, or BK virus) or JCpolyomavirus (JCV, JC PyV, or JC virus).

In some embodiments, the severity of the infection is reduced with thetreatment of the compounds. The reduction of the severity of theinfection can be measured by the reduction of the symptoms which presentwith the infection.

The compounds of the present disclosure have sialidase activity. In someinstances, the compounds having sialidase activity are a fusion proteinin which the portion having sialidase activity is fused to a protein orprotein fragment not having sialidase activity. In some instances theportion having sialidase activity is fused to an anchoring domain. Insome instances the anchoring domain is GAG.

DAS181 (SEQ ID NOs: 13 and 14) is a fusion protein compound comprisingthe catalytic domain of a sialidase (A. viscous) and an anchoring domainthat is a human amphiregulin GAG-binding domain. In some instances ofthe present disclosure, DAS181 could be used to treat the viralinfection and disorders associated therewith (e.g., late onsethaemorrhagic cystitis, BKV nephropathy, or ureteral stenosis, caused byBK virus; progressive multifocal leukoencephalopathy, granule cellneuronopathy, encephalopathy, meningitis, or immune reconstitutioninflammatory syndrome, caused by JC virus).

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Generally, the nomenclatureused herein and the manufacture or laboratory procedures described beloware well known and commonly employed in the art. Conventional methodsare used for these procedures, such as those provided in the art andvarious general references. Where a term is provided in the singular,the inventors also contemplate the plural of that term. Where there arediscrepancies in terms and definitions used in references that areincorporated by reference, the terms used in this application shall havethe definitions given herein. As employed throughout the disclosure, thefollowing terms, unless otherwise indicated, shall be understood to havethe following meanings:

A “target cell” is any cell that can be infected by the viral pathogen,e.g., a kidney cell that can be infected by a BK virus; oroligodendrocytes, astrocytes, lymphocytes, kidney epithelium cells,tonsil stromal cells, or plasma cells that can be infected by a JCvirus.

A “domain that can anchor said at least one sialidase domain to themembrane of a target cell”, also called an “extracellular anchoringdomain” or simply, “anchoring domain” refers to a moiety that caninteract with a moiety that is at or on the exterior of a cell surfaceor is in close proximity to the surface of a cell. An extracellularanchoring domain can be reversibly or irreversibly linked to one or moremoieties, such as, preferably, one or more sialidase domains, andthereby cause the one or more attached therapeutic moieties to beretained at or in close proximity to the exterior surface of aeukaryotic cell. Preferably, an extracellular anchoring domain interactswith at least one molecule on the surface of a target cell or at leastone molecule found in close association with the surface of a targetcell. For example, an extracellular anchoring domain can bind a moleculecovalently or noncovalently associated with the cell membrane of atarget cell, or can bind a molecule present in the extracellular matrixsurrounding a target cell. An extracellular anchoring domain preferablyis a peptide, polypeptide, or protein, and can also comprise anyadditional type of chemical entity, including one or more additionalproteins, polypeptides, or peptides, a nucleic acid, peptide nucleicacid, nucleic acid analogue, nucleotide, nucleotide analogue, smallorganic molecule, polymer, lipids, steroid, fatty acid, carbohydrate, ora combination of any of these.

As used herein, a protein or peptide sequences is “substantiallyhomologous” to a reference sequence when it is either identical to areference sequence, or comprises one or more amino acid deletions, oneor more additional amino acids, or more one or more conservative aminoacid substitutions, and retains the same or essentially the sameactivity as the reference sequence. Conservative substitutions may bedefined as exchanges within one of the following five groups:

I. Small, aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr,Pro, Gly

II. Polar, negatively charged residues and their amides: Asp, Asn, Glu,Gln

III. Polar, positively charged residues: His, Arg, Lys

IV. Large, aliphatic nonpolar residues: Met, Leu, Ile, Val, Cys

V. Large aromatic residues: Phe, Try, Trp

Within the foregoing groups, the following substitution are consideredto be “highly conservative”: Asp/Glu, His/Arg/Lys, Phe/Tyr/Trp, andMet/Leu/Ile/Val. Semi-conservative substitutions are defined to beexchanges between two of groups (I)-(V) above which are limited tosupergroup (A), comprising (I), (II), and (III) above, or to supergroup(B), comprising (IV) and (V) above. In addition, where hydrophobic aminoacids are specified in the application, they refer to the amino acidsAla, Gly, Pro, Met, Leu, Ile, Val, Cys, Phe, and Trp, whereashydrophilic amino acids refer to Ser, Thr, Asp, Asn, Glu, Gln, His, Arg,Lys, and Tyr.

As used herein, the phrase “therapeutically effective amount” refers tothe amounts of active compounds or their combination that elicit thebiological or medicinal response that is being sought in a tissue,system, animal, individual or human by a researcher, veterinarian,medical doctor or other clinician, which includes one or more of thefollowing:

(1) inhibiting the disease and its progression; for example, inhibitinga disease, condition or disorder in an individual who is experiencing ordisplaying the pathology or symptomatology of the disease, condition ordisorder (i.e., arresting further development of the pathology and/orsymptomatology) such as in the case of BK or JC virus infection, and

(2) ameliorating the disease; for example, ameliorating a disease,condition or disorder in an individual who is experiencing or displayingthe pathology or symptomatology of the disease, condition or disorder(i.e., reversing the pathology and/or symptomatology) such as in thecase of BK or JC virus infection.

As used herein, the phrase “treating (including treatment)” includes oneor more of the following:

(1) inhibiting the disease and its progression; for example, inhibitinga disease, condition or disorder in an individual who is experiencing ordisplaying the pathology or symptomatology of the disease, condition ordisorder (i.e., arresting further development of the pathology and/orsymptomatology), and

(2) ameliorating the disease; for example, ameliorating a disease,condition or disorder in an individual who is experiencing or displayingthe pathology or symptomatology of the disease, condition or disorder.

A “sialidase” is an enzyme that can remove a sialic acid residue from asubstrate molecule. The sialidases (N-acylneuraminosylglycohydrolases,EC 3.2.1.18) are a group of enzymes that hydrolytically remove sialicacid residues from sialo-glycoconjugates. Sialic acids are alpha-ketoacids with 9-carbon backbones that are usually found at the outermostpositions of the oligosaccharide chains that are attached toglycoproteins and glycolipids. One of the major types of sialic acids isN-acetylneuraminic acid (NeuSAc), which is the biosynthetic precursorfor most of the other types. The substrate molecule can be, asnonlimiting examples, an oligosaccharide, a polysaccharide, aglycoprotein, a ganglioside, or a synthetic molecule. For example, asialidase can cleave bonds having alpha (2,3)-Gal, alpha(2,6)-Gal, oralpha (2,8)-Gal linkages between a sialic acid residue and the remainderof a substrate molecule. A sialidase can also cleave any or all of thelinkages between the sialic acid residue and the remainder of thesubstrate molecule. Two major linkages between NeuSAc and thepenultimate galactose residues of carbohydrate side chains are found innature, NeuSAc alpha (2,3)-Gal and NeuSAc alpha (2,6)-Gal. Both NeuSAcalpha (2,3)-Gal and NeuSAc alpha (2,6)-Gal molecules can be recognizedby influenza viruses as the receptor, although human viruses seem toprefer NeuSAc alpha (2,6)-Gal, avian and equine viruses predominantlyrecognize NeuSAc alpha (2,3)Gal. A sialidase can be anaturally-occurring sialidase, an engineered sialidase (such as, but notlimited to a sialidase whose amino acid sequence is based on thesequence of a naturally-occurring sialidase, including a sequence thatis substantially homologous to the sequence of a naturally-occurringsialidase). As used herein, “sialidase” can also mean the active portionof a naturally-occurring sialidase, or a peptide or protein thatcomprises sequences based on the active portion of a naturally-occurringsialidase.

A “fusion protein” is a protein comprising amino acid sequences from atleast two different sources. A fusion protein can comprise amino acidsequence that is derived from a naturally occurring protein or issubstantially homologous to all or a portion of a naturally occurringprotein, and in addition can comprise from one to a very large number ofamino acids that are derived from or substantially homologous to all ora portion of a different naturally occurring protein. In thealternative, a fusion protein can comprise amino acid sequence that isderived from a naturally occurring protein or is substantiallyhomologous to all or a portion of a naturally occurring protein, and inaddition can comprise from one to a very large number of amino acidsthat are synthetic sequences.

A “sialidase catalytic domain protein” is a protein that comprises thecatalytic domain of a sialidase, or an amino acid sequence that issubstantially homologous to the catalytic domain of a sialidase, butdoes not comprises the entire amino acid sequence of the sialidase thecatalytic domain is derived from, wherein the sialidase catalytic domainprotein retains substantially the same activity as the intact sialidasethe catalytic domain is derived from. A sialidase catalytic domainprotein can comprise amino acid sequences that are not derived from asialidase, but this is not required. A sialidase catalytic domainprotein can comprise amino acid sequences that are derived from orsubstantially homologous to amino acid sequences of one or more otherknown proteins, or can comprise one or more amino acids that are notderived from or substantially homologous to amino acid sequences ofother known proteins.

I. Composition for Preventing or Treating Infection by a Pathogen

The present disclosure relates to compounds (agents) that include apeptide. The compounds include all or a catalytic portion of asialidase. In some cases the compound includes at least one domain thatcan associate the sialidase or portion thereof with a eukaryotic cell.By “peptide or protein-based” compounds, it is meant that a compoundthat includes a portion having an amino acid framework, in which theamino acids are joined by peptide bonds. A peptide or protein-basedcompound can also have other chemical compounds or groups attached tothe amino acid framework or backbone, including moieties that contributeto the anchoring activity of the anchoring domain, or moieties thatcontribute to the infection-preventing activity or the sialidase domain.For example, the protein-based therapeutics of the present disclosurecan comprise compounds and molecules such as but not limited to:carbohydrates, fatty acids, lipids, steroids, nucleotides, nucleotideanalogues, nucleic acid molecules, nucleic acid analogues, peptidenucleic acid molecules, small organic molecules, or even polymers. Theprotein-based therapeutics of the present disclosure can also comprisemodified or non-naturally occurring amino acids. Non-amino acid portionsof the compounds can serve any purpose, including but not limited to:facilitating the purification of the compound, improving the solubilityor distribution or the compound (such as in a therapeutic formulation),linking domains of the compound or linking chemical moieties to thecompound, contributing to the two dimensional or three-dimensionalstructure of the compound, increasing the overall size of the compound,increasing the stability of the compound, and contributing to theanchoring activity or therapeutic activity of the compound.

The peptide or protein-based compounds of the present disclosure canalso include protein or peptide sequences in addition to those thatcomprise anchoring domains or sialidase domains. The additional proteinsequences can serve any purpose, including but not limited to any of thepurposes outlined above (facilitating the purification of the compound,improving the solubility or distribution or the compound, linkingdomains of the compound or linking chemical moieties to the compound,contributing to the two-dimensional or three-dimensional structure ofthe compound, increasing the overall size of the compound, increasingthe stability of the compound, or contributing to the anchoring activityor therapeutic activity of the compound). Preferably any additionalprotein or amino acid sequences are part of a single polypeptide orprotein chain that includes the sialidase domain or domains, but anyfeasible arrangement of protein sequences is within the scope of thepresent disclosure.

The anchoring domain and sialidase domain can be arranged in anyappropriate way that allows the compound to bind at or near a targetcell membrane such that the therapeutic sialidase can exhibit anextracellular activity that prevents or impedes infection of the targetcell by a pathogen. The compound will preferably have at least oneprotein or peptide-based anchoring domain and at least one peptide orprotein-based sialidase domain. In this case, the domains can bearranged linearly along the peptide backbone in any order. The anchoringdomain can be N-terminal to the sialidase domain, or can be C-terminalto the sialidase domain.

It is also possible to have one or more sialidase domains flanked by atleast one anchoring domain on each end. Alternatively, one or moreanchoring domains can be flanked by at least one sialidase domain oneach end. Chemical, or preferably, peptide, linkers can optionally beused to join some or all of the domains of a compound. It is alsopossible to have the domains in a nonlinear, branched arrangement. Forexample, the sialidase domain can be attached to a derivatized sidechain of an amino acid that is part of a polypeptide chain that alsoincludes, or is linked to, the anchoring domain.

A compound of the present disclosure can have more than one anchoringdomain. In cases in which a compound has more than one anchoring domain,the anchoring domains can be the same or different. A compound of thepresent disclosure can have more than one sialidase domain. In cases inwhich a compound has more than one sialidase domain, the sialidasedomains can be the same or different. Where a compound comprisesmultiple anchoring domains, the anchoring domains can be arranged intandem (with or without linkers) or on alternate sides of other domains,such as sialidase domains. Where a compound comprises multiple sialidasedomains, the sialidase domains can be arranged in tandem (with orwithout linkers) or on alternate sides of other domains, such as, butnot limited to, anchoring domains.

A peptide or protein-based compound of the present disclosure can bemade by any appropriate way, including purifying naturally occurringproteins, optionally proteolytically cleaving the proteins to obtain thedesired functional domains, and conjugating the functional domains toother functional domains. Peptides can also be chemically synthesized,and optionally chemically conjugated to other peptides or chemicalmoieties. Preferably, however, a peptide or protein-based compound ofthe present disclosure is made by engineering a nucleic acid constructto encode at least one anchoring domain and at least one sialidasedomain together (with or without nucleic acid linkers) in a continuouspolypeptide. The nucleic acid constructs, preferably having appropriateexpression sequences, can be transfected into prokaryotic or eukaryoticcells, and the therapeutic protein-based compound can be expressed bythe cells and purified. Any desired chemical moieties can optionally beconjugated to the peptide or protein-based compound after purification.In some cases, cell lines can be chosen for expressing the protein-basedtherapeutic for their ability to perform desirable post-translationalmodifications (such as, but not limited to glycosylation).

A great variety of constructs can be designed and their protein productstested for desirable activities (such as, for example, binding activityof an anchoring domain or catalytic activity of a sialidase domain). Theprotein products of nucleic acid constructs can also be tested for theirefficacy in preventing or impeding infection of a target cell by apathogen. In vitro and in vivo tests for the infectivity of pathogensare known in the art.

Anchoring Domain

As used herein, an “extracellular anchoring domain” or “anchoringdomain” is any moiety that interact with an entity that is at or on theexterior surface of a target cell or is in close proximity to theexterior surface of a target cell. An anchoring domain serves to retaina compound of the present disclosure at or near the external surface ofa target cell. An extracellular anchoring domain preferably binds 1) amolecule expressed on the surface of a target cell, or a moiety, domain,or epitope of a molecule expressed on the surface of a target cell, 2) achemical entity attached to a molecule expressed on the surface of atarget cell, or 3) a molecule of the extracellular matrix surrounding atarget cell.

An anchoring domain is preferably a peptide or protein domain (includinga modified or derivatized peptide or protein domain), or comprises amoiety coupled to a peptide or protein. A moiety coupled to a peptide orprotein can be any type of molecule that can contribute to theinteraction of the anchoring domain to an entity at or near the targetcell surface, and is preferably an organic molecule, such as, forexample, nucleic acid, peptide nucleic acid, nucleic acid analogue,nucleotide, nucleotide analogue, small organic molecule, polymer,lipids, steroid, fatty acid, carbohydrate, or any combination of any ofthese.

Target tissue or target cell type includes the sites in an animal orhuman body where a pathogen invades or amplifies. For example, a targetcell can be a kidney cell that can be infected by a BK virus; or aneuronal cell that can be infected by a JC virus. A compound or agentsof the present disclosure can comprise an anchoring domain that caninteract with a cell surface entity, for example, that is specific forthe target cell type.

A compound for treating infection by a pathogen can comprise ananchoring domain that can bind at or near the surface of a target cell.For example, heparin/sulfate, closely related to heparin, is a type ofGAG that is ubiquitously present on cell membranes, including thesurface of respiratory epithelium. Many proteins specifically bind toheparin/heparan sulfate, and the GAG-binding sequences in these proteinshave been identified (Meyer, F A, King, M and Gelman, R A. (1975)Biochimica et BiophysicaActa 392: 223-232; Schauer, S. ed., pp 233.Sialic Acids Chemistry, Metabolism and Function. Springer-Verlag, 1982).For example, the GAG-binding sequences of human platelet factor 4 (PF4)(SEQ ID NO:2), human interleukin 8 (IL8) (SEQ ID NO:3),humanantithrombin III (AT III) (SEQ ID NO:4), human apoprotein E (ApoE)(SEQ ID NO:5), human angio-associated migratory cell protein (AAMP) (SEQID NO:6), or human amphiregulin (SEQ ID NO:7) have been shown to havevery high affinity (in the nanomolar range) towards heparin (Lee, M Kand Lander, A D. (1991) Pro Natl Acad Sci USA 88:2768-2772; Goger, B,Halden, Y, Rek, A, Mosl, R, Pye, D. Gallagher, J and Kungl, A J. (2002)Biochem. 41:1640-1646; Witt, D P and Lander A D (1994) Curr Bio4:394-400; Weisgraber, K H, Rail, S C, Mahley, R W, Milne, R W andMarcel, Y. (1986) J Bio Chem 261:2068-2076). These sequences, or othersequences that have been identified or are identified in the future asheparin/heparan sulfate binding sequences, or sequences substantiallyhomologous to identified heparin/heparan sulfate binding sequences thathave heparin/heparan sulfate binding activity, can be used asepithelium-anchoring-domains in compounds of the present disclosure thatcan be used.

Sialidase Domain

A sialidase that can cleave more than one type of linkage between asialic acid residue and the remainder of a substrate molecule, inparticular, a sialidase that can cleave both α(2,6)-Gal and α(2,3)-Gallinkages can be used in the compounds of the disclosure. Sialidasesinclude are the large bacterial sialidases that can degrade the receptorsialic acids Neu5Ac alpha(2,6)-Gal and Neu5Ac alpha(2,3)-Gal. Forexample, the bacterial sialidase enzymes from Clostridium perfringens(Genbank Accession Number X87369), Actinomyces viscosus, Arthrobacterureafaciens, or Micromonospora viridifaciens (Genbank Accession NumberD01045) can be used. Sialidase domains of compounds of the presentdisclosure can comprise all or a portion of the amino acid sequence of alarge bacterial sialidase or can comprise amino acid sequences that aresubstantially homologous to all or a portion of the amino acid sequenceof a large bacterial sialidase. In one preferred embodiment, a sialidasedomain comprises a sialidase encoded by Actinomyces viscosus, such asthat of SEQ ID NO: 12, or such as sialidase sequence substantiallyhomologous to SEQ ID NO: 12. In yet another preferred embodiment, asialidase domain comprises the catalytic domain of the Actinomycesviscosus sialidase extending from amino acids 274-666 of SEQ ID NO:12,or a substantially homologous sequence.

Additional sialidases include the human sialidases such as those encodedby the genes NEU2 (SEQ ID NO:8; Genbank Accession Number Y16535; Monti,E, Preti, Rossi, E., Ballabio, A and Borsani G. (1999) Genomics57:137-143) and NEU4 (SEQ ID NO:9; Genbank Accession Number NM080741;Monti, E, Preti, A, Venerando, Band Borsani, G. (2002) Neurochem Res27:646-663). Sialidase domains of compounds of the present disclosurecan comprise all or a portion of the amino acid sequences of a sialidaseor can comprise amino acid sequences that are substantially homologousto all or a portion of the amino acid sequences of a sialidase.Preferably, where a sialidase domain comprises a portion of the aminoacid sequences of a naturally occurring sialidase, or sequencessubstantially homologous to a portion of the amino acid sequences of anaturally occurring sialidase, the portion comprises essentially thesame activity as the intact sialidase. The present disclosure alsoincludes sialidase catalytic domain proteins. As used herein a“sialidase catalytic domain protein” comprises a catalytic domain of asialidase but does not comprise the entire amino acid sequence of thesialidase from which the catalytic domain is derived. A sialidasecatalytic domain protein has sialidase activity. Preferably, a sialidasecatalytic domain protein comprises at least 10%, at least 20%, at least50%, at least 70% of the activity of the sialidase from which thecatalytic domain sequence is derived. More preferably, a sialidasecatalytic domain protein comprises at least 90% of the activity of thesialidase from which the catalytic domain sequence is derived.

A sialidase catalytic domain protein can include other amino acidsequences, such as but not limited to additional sialidase sequences,sequences derived from other proteins, or sequences that are not derivedfrom sequences of naturally occurring proteins. Additional amino acidsequences can perform any of a number of functions, includingcontributing other activities to the catalytic domain protein, enhancingthe expression, processing, folding, or stability of the sialidasecatalytic domain protein, or even providing a desirable size or spacingof the protein.

A preferred sialidase catalytic domain protein is a protein thatcomprises the catalytic domain of the A. viscosus sialidase. Preferably,an A. viscosus sialidase catalytic domain protein comprises amino acids270-666 of the A. viscosus sialidase sequence (SEQ ID NO:12).Preferably, an A. Viscosus sialidase catalytic domain protein comprisesan amino acid sequence that begins at any of the amino acids from aminoacid 270 to amino acid 290 of the A. viscosus sialidase sequence (SEQ IDNO: 12) and ends at any of the amino acids from amino acid 665 to aminoacid 901 of said A. viscosus sialidase sequence (SEQ ID NO: 12), andlacks any A. viscosus sialidase protein sequence extending from aminoacid 1 to amino acid 269. (As used herein “lacks any A. viscosussialidase protein sequence extending from amino acid 1 to amino acid269” means lacks any stretch of four or more consecutive amino acids asthey appear in the designated protein or amino acid sequence.)

In some preferred embodiments, an A. viscosus sialidase catalytic domainprotein comprises amino acids 274-681 of the A. viscosus sialidasesequence (SEQ ID NO: 12) and lacks other A. viscosus sialidase sequence.In some preferred embodiments, an A. viscosus sialidase catalytic domainprotein comprises amino acids 274-666 of the A. viscosus sialidasesequence (SEQ ID NO: 12) and lacks any other A. viscosus sialidasesequence. In some preferred embodiments, an A. viscosus sialidasecatalytic domain protein comprises amino acids 290-666 of the A.viscosus sialidase sequence (SEQ ID NO: 12) and lacks any other A.viscosus sialidase sequence. In yet other preferred embodiments, an A.viscosus sialidase catalytic domain protein comprises amino acids290-681 of the A. viscosus sialidase sequence (SEQ ID NO: 12) and lacksany other A. viscosus sialidase sequence.

Linkers

A compound of the present disclosure can optionally include one or morelinkers that can join domains of the compound. Linkers can be used toprovide optimal spacing or folding of the domains of a compound. Thedomains of a compound joined by linkers can be sialidase domains,anchoring domains, or any other domains or moieties of the compound thatprovide additional functions such as enhancing compound stability,facilitating purification, etc. A linker used to join domains ofcompounds of the present disclosure can be a chemical linker or an aminoacid or peptide linker. Where a compound comprises more than one linker,the linkers can be the same or different. Where a compound comprisesmore than one linker, the linkers can be of the same or differentlengths.

Many chemical linkers of various compositions, polarity, reactivity,length, flexibility, and cleavability are known in the art of organicchemistry. Preferred linkers of the present disclosure include aminoacid or peptide linkers. Peptide linkers are well known in the art.Preferably linkers are between one and one hundred amino acids inlength, and more preferably between one and thirty amino acids inlength, although length is not a limitation in the linkers of thecompounds of the present disclosure. Preferably linkers comprise aminoacid sequences that do not interfere with the conformation and activityof peptides or proteins encoded by monomers of the present disclosure.Some preferred linkers of the present disclosure are those that includethe amino acid glycine. For example, linkers having the sequence: (GGGGS(SEQ ID NO:10))n, where n is a whole number between 1 and 20, or morepreferably between 1 and 12, can be used to link domains of therapeuticcompounds of the present disclosure.

The present disclosure also includes nucleic acid molecules that encodeprotein-based compounds of the present disclosure that comprise at leastone sialidase domain and at least one anchoring domain. The nucleic acidmolecules can have codons optimized for expression in particular celltypes, such as, for example E. coli or human cells. The nucleic acidmolecules or the present disclosure that encode protein-based compoundsof the present disclosure that comprise at least one sialidase domainand at least one anchoring domain can also comprise other nucleic acidsequences, including but not limited to sequences that enhance geneexpression. The nucleic acid molecules can be in vectors, such as butnot limited to expression vectors.

Administration

The compound is administered so that it comes into contact with thetarget cells, but is preferably not administered systemically to thepatient. For example, in the case of BK virus infection of the kidney, acomposition comprising a sialidase (e.g., a composition comprisingDAS181) can be infused into the kidney. In the case of JC virusinfection of the nervous system, a composition comprising a sialidase(e.g., a composition comprising DAS181) can be injected into the brain,spinal cord, or cerebrospinal fluid.

Nucleic Acid Molecules

The present disclosure also comprises nucleic acid molecules that encodeprotein-based compounds of the present disclosure that comprise acatalytic domain of a sialidase. The nucleic acid molecules can havecodons optimized for expression in particular cell types, such as, forexample E. coli or human cells. The nucleic acid molecules or thepresent disclosure that encode protein-based compounds of the presentdisclosure that comprise at least one catalytic domain of a sialidasecan also comprise other nucleic acid sequences, including but notlimited to sequences that enhance gene expression. The nucleic acidmolecules can be in vectors, such as but not limited to expressionvectors.

II. Pharmaceutical Compositions

The present disclosure includes compounds of the present disclosureformulated as pharmaceutical compositions. The pharmaceuticalcompositions comprise a pharmaceutically acceptable carrier prepared forstorage and preferably subsequent administration, which have apharmaceutically effective amount of the compound in a pharmaceuticallyacceptable carrier or diluent. Acceptable carriers or diluents fortherapeutic use are well known in the pharmaceutical art, and aredescribed, for example, in Remington's Pharmaceutical Sciences, 18thEd., Mack Publishing Co., Easton, Pa. (1990)). Preservatives,stabilizers, dyes and even flavoring agents can be provided in thepharmaceutical composition. For example, sodium benzoate, sorbic acidand esters of p-hydroxybenzoic acid can be added as preservatives. Inaddition, antioxidants and suspending agents can be used.

The pharmaceutically effective amount of a test compound required as adose will depend on the route of administration, the type of animal orpatient being treated, and the physical characteristics of the specificanimal under consideration. The dose can be tailored to achieve adesired effect, but will depend on such factors as weight, diet,concurrent medication and other factors which those skilled in themedical arts will recognize. In practicing the methods of the presentdisclosure, the pharmaceutical compositions can be used alone or incombination with one another, or in combination with other therapeuticor diagnostic agents. These products can be utilized in vivo, preferablyin a mammalian patient, preferably in a human, or in vitro. In employingthem in vivo, the pharmaceutical compositions can be administered to thepatient in a variety of ways, preferably topically to the target cells,topically to the locus of infection or topically to tissue comprisingthe target cells.

Accordingly, in some embodiments, the methods comprise administration ofthe agent and a pharmaceutically acceptable carrier. In someembodiments, the ophthalmic composition is a liquid composition,semi-solid composition, insert, film, microparticles or nanoparticles.

III. Method of Treating an Infection by a Pathogen

The method of the present disclosure includes: treating a subject thatis infected with a pathogen or at risk of being infected with a pathogenwith a pharmaceutical composition of the present disclosure thatcomprises a protein-based compound that comprises a sialidase activity.In some preferred embodiments the method includes applying atherapeutically effective amount of a pharmaceutical composition of thepresent disclosure to target cells of a subject. The sialidase activitycan be an isolated naturally occurring sialidase protein, or arecombinant protein substantially homologous to at least a portion of anaturally occurring sialidase. A preferred pharmaceutical compositioncomprises a sialidase with substantial homology to the A. viscosussialidase (SEQ ID NO:12). The subject to be treated can be an animal orhuman subject. In yet another aspect, the method includes: treating asubject that is infected with a pathogen with a pharmaceuticalcomposition of the present disclosure that comprises a protein-basedcompound that comprises a sialidase catalytic domain. In some preferredembodiments, the method includes applying a therapeutically effectiveamount of a pharmaceutical composition of the present disclosure toepithelial cells of a subject. The sialidase catalytic domain ispreferably substantially homologous to the catalytic domain of anaturally occurring sialidase. A preferred pharmaceutical compositioncomprises a sialidase catalytic domain with substantial homology toamino acids 274-666 the A. viscosus sialidase (SEQ ID NO: 12). Thesubject to be treated can be an animal or human subject. In some casesthe compound is DAS181.

Dosage

As will be readily apparent to one skilled in the art, the useful invivo dosage to be administered and the particular mode of administrationwill vary depending upon the age, weight and type of patient beingtreated, the particular pharmaceutical composition employed, and thespecific use for which the pharmaceutical composition is employed. Thedetermination of effective dosage levels, that is the dose levelsnecessary to achieve the desired result, can be accomplished by oneskilled in the art using routine methods as discussed above. Innon-human animal studies, applications of the pharmaceuticalcompositions are commenced at higher dose levels, with the dosage beingdecreased until the desired effect is no longer achieved or adverse sideeffects are reduced or disappear. The dosage for a compound of thepresent disclosure can range broadly depending upon the desired affects,the therapeutic indication, route of administration and purity andactivity of the compound. Typically, human clinical applications ofproducts are commenced at lower dosage levels, with dosage level beingincreased until the desired effect is achieved. Alternatively,acceptable in vitro studies can be used to establish useful doses androutes of administration of the test compound. Typically, dosages can bebetween about 1 ng/kg and about 10 mg/kg, preferably between about 10ng/kg and about 1 mg/kg, and more preferably between about 100 ng/kg andabout 100 micrograms/kg.

In one preferred regimen, appropriate dosages are administered to eachpatient by infusion or injection directly to the infected organ. It willbe understood, however, that the specific dose level and frequency ofdosage for any particular patient maybe varied and will depend upon avariety of factors including the activity of the specific salt or otherform employed, the metabolic stability and length of action of thatcompound, the age of the patient, body weight of the patient, generalhealth of the patient, sex of the patient, diet of the patient, mode andtime of administration, rate of excretion, drug combination, theseverity of the particular condition, and the host undergoing therapy.

EXAMPLES Example 1: Preparation of a Suspension of DAS181 Microparticlesfor Use in Treating Infections

Purification of DAS181

DAS181 is a fusion protein containing the heparin (glycosaminoglycan, orGAG) binding domain from human amphiregulin fused via its N-terminus tothe C-terminus of a catalytic domain of Actinomyces Viscosus (e.g.,sequence of amino acids set forth in SEQ ID NO: 13 (no amino terminalmethionine) and SEQ ID NO: 14 (including amino terminal methionine). TheDAS181 protein used in the examples below was purified as described inMalakhov et al., Antimicrob. Agents Chemother., 1470-1479 (2006), whichis incorporated in its entirety by reference herein. Briefly, the DNAfragment coding for DAS181 was cloned into the plasmid vector pTrc99a(Pharmacia) under the control of an IPTG(isopropyl-β-D-thiogalactopyranoside)-inducible promoter. The resultingconstruct was expressed in the BL21 strain of Escherichia Coli (E.Coli). The E. coli cells expressing the DAS181 protein were washed bydiafiltration in a fermentation harvest wash step using Toyopearl buffer1, UFP-500-E55 hollow fiber cartridge (GE Healthcare) and aWatson-Marlow peristaltic pump. The recombinant DAS181 protein was thenpurified in bulk from the cells as described in US 20050004020 and US20080075708, which are incorporated in their entirety by referenceherein.

Activity of DAS181

The sialidase activity of DAS181 was measured using the fluorogenicsubstrate 4-methylumbelliferyl-N-acetyl-α-D-neuraminic acid (4-MU-NANA;Sigma). One unit of sialidase is defined as the amount of enzyme thatreleases 10 nmol of MU from 4-MU-NANA in 10 minutes at 37° C. (50 mMCH₃COOH—NaOH buffer, pH 5.5) in a reaction that contains 20 nmol of4-MU-NANA in a 0.2 ml volume (Potier et al., Anal. Biochem., 94:287-296,1979). The specific activity of DAS181 was determined to be 1,300 U/mgprotein (0.77 μg DAS181 protein per unit of activity).

Microparticle Preparation

The following ingredients were then combined to form DAS181microparticles in a large scale batch process:

-   -   (a) 75 mg/ml Histidine, 0.107M citric acid, pH 5.0 and 1M        Trehalose stock solutions were sterile filtered into and        combined in an Excipient Bottle.    -   (b) The contents of the Excipient Bottle were added, with        mixing, to a Compounding Vessel containing 125 mg/ml DAS181        protein prepared as described in Example 1.    -   (c) Isopropanol was sterile filtered into an Isopropanol Bag    -   (d) The content of the Isopropanol Bag was pumped into the        Compounding Vessel while mixing vigorously to form the Feedstock        Solution. The final composition of the Feedstock Solution was as        follows: 70 mg/ml DAS181, 26% isopropanol, 9.8 mg/ml histidine,        9.8 mg/ml trehalose, 2.69 mg/ml citric acid, pH 5.0. The time        between initiating the addition of isopropanol and starting the        lyophilization cycle was between 90 minutes and 120 minutes    -   (e) Stainless Steel trays that had undergone depyrogenation were        each filled with 950 g of the Feedstock Solution, using a        metering pump    -   (f) The filled Stainless Steel trays were subjected to a        Lyophilization Cycle as follows:        -   a. the feedstock solution in the lyophilization trays were            gasketed and placed in the lyophilizer shelves at 25° C. for            5 minutes;        -   b. the temperature of the shelves was lowered to −55° C. at            a ramp rate of −0.4° C./minute;        -   c. the trays were held at −55° C. for between 60 and 180            minutes;        -   d. primary drying was accomplished by setting the condenser            to <−60° C., applying a vacuum of 125 mTorr with 250 mTorr            dead band and increasing the temperature to −40° C. at a            ramp rate of 0.125° C./minute and further to a temperature            of −30° C. at 0.167° C./minute;        -   e. the temperature was held at −30° C. for between 5000 and            6500 minutes;        -   f. secondary drying was accomplished by increasing the            temperature to 15° C. at a ramp rate of 0.5° C./minute,            holding at 15° C. for 30 minutes, then further ramping up to            a temperature of 30° C. at a ramp rate of 0.5° C./minute;        -   g. the temperature was held at 30° C. for between 300 and            500 minutes; and        -   h. the vacuum was released and the lyophilizer was            backfilled with nitrogen to prevent oxidation of the            microparticle formulations before transferring into bottles            for bulk mixing and aliquoting the bulk powder for storage            at <−15° C.

Physical Parameters:

The DAS181 dry powder microparticles prepared according to the abovemethod have a mass median aerodynamic diameter (MMAD) of about 10microns and a GSD of between 1 and 2.

Suspension of Microparticles

To prepare 1 ml of a 100 mg DAS181/ml suspension, 125 mg ofmicroparticles prepared as described were placed in a vial in acontrolled RH environment (typically 10-30% RH). Next, 450 μL of PEG 300was added to the vial and gently mixed with the microparticles. Themixture was held for 5 minutes to allow the microparticles to interactwith the PEG 300. Next, 4504 of water is added to the vial and thecontents are gently mixed for 2-3 minutes or until a homogeneoussuspension is achieved.

Injectability was measured using a NE-1010 syringe pump with a DPM-3digital mount meter attached to the plunger rail. Standard 1 mL BDsyringes are used with 27G×½ PrecisionGlide BD needles. Injectabilityvalues are reported in unit of lbs of force measured. Viscosity wasmeasured using a Brookfield DV-1 Prime with a CPE-44PY cup and a CPE-40cone spindle. Injection force of less than 50N is considered asinjectable. The conversion unit of lbs to N is 1 lbs=4.4 N.

The above method produced suspensions with good injectability. Goodresults were obtained when the ratio of PEG 300 to water was: 50:50,65:35 and 75:25. When PEG 200 was used, good results were obtained whenthe ratio of PEG 300 to water was 65:35 and 75:25.

In addition to polyethylene glycol (PEG 200, PEG 300, PEG 400, PEG 500,PEG 600), polysorbate 80, polysorbate 20 (Polyoxyethylene (20) sorbitanmonooleate), propylene glycol, thioglycerol, tricaprylin, triolein, andversetamide are useful first media for adding to the proteinmicroparticles.

The second media is water that can include salts, buffers, preservativesand other pharmaceutically acceptable excipients.

Example 2: Inhibition of BK Polyomavirus Infection by DAS181

CCD1105 cells were plated in 8-well chamber slides at 2,500 cells perwell, incubated overnight at 37° C. The cells were then treated withvarious concentrations of DAS181 for 24 hours at 37° C. Concentrationsof DAS181 used include: (A) 0 nM, (B) 0.1 nM, (C) 1 nM, (D) 10 nM, (E)100 nM, and (F) 1000 nM. After 24 hours, DAS181 containing media wasaspirated, cells were washed with PBS, and infected with BK virus at anMOI of 0.1 for 3 hours at 37° C. After incubation with virus, cells werewashed with PBS followed by addition of media with or without DAS181 toeach well and changed every 24 hours. Four days post infection cellswere fixed and stained for VP1 protein by immunofluorescent staining;nuclei were counterstained with DAPI. As shown in FIG. 1, 0.1 nM ofDAS181 effectively reduced the BK infection of CCD1105 cells by 50%; 100nm of DAS181 reduced BK infection of CCD1105 cells by about 66%, and1000 nm of DAS181 reduced BK infection of CCD1105 cells by about 77%.

The invention claimed is:
 1. A method of treating an infection by a BKpolyomavirus or a BK polyomavirus-associated disorder in a subject, themethod comprising administering to the subject an effective amount of apolypeptide comprising the amino acid sequence of SEQ ID NO: 13 or SEQID NO:14.
 2. A method of reducing the risk or severity of an infectionby BK polyomavirus or a BK polyomavirus-associated disorder in asubject, the method comprising administering to the subject an effectiveamount of a polypeptide comprising the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:14.
 3. The method of claim 1, comprising administering acomposition comprising microparticles comprising a polypeptidecomprising the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO:14. 4.The method of claim 1, wherein the agent is not systemicallyadministered.
 5. The method of claim 1, wherein the subject isimmunocompromised.
 6. The method of claim 1, wherein the infection isassociated with an event selected from the group consisting of: an HIVinfection, cessation of immunosuppressive therapy, commencement ofantiviral therapy, and monoclonal antibody autoimmune disease therapy.7. The method of claim 1, wherein the subject is a transplant patientundergoing immunosuppressive therapy.
 8. The method of claim 1, whereinthe subject is being treated with an immunomodulatory agent that reducesone or more aspects of immune function.
 9. The method of claim 8,wherein the immunomodulatory agent is selected from the group consistingof natalizumab, rituximab, efalizumab and infliximab.
 10. The method ofclaim 1, wherein the polyomavirus binds a sialic acid-containingcomponent on the surface of a target cell.
 11. The method of claim 1,wherein the subject has undergone haematopoietic stem cell transplant oris being prepared for haematopoietic stem cell transplant.
 12. Themethod of claim 1, wherein the disorder is BKV nephropathy.
 13. Themethod of claim 1, wherein the disorder is nephritis.
 14. The method ofclaim 1, wherein the disorder is hemorrhagic cystitis.
 15. The method ofclaim 1, wherein the disorder is ureteral stenosis.
 16. The method ofclaim 1, wherein the subject has undergone solid organ transplant or isbeing treated in preparation for solid organ transplant.
 17. The methodof claim 1, wherein the disorder is lupus.
 18. The method of claim 1,wherein the agent is administered to the kidneys.
 19. The method ofclaim 1, wherein the agent is administered to the ureter.
 20. The methodof claim 1, wherein the agent is administered to the bladder.
 21. Themethod of claim 1, wherein the agent is administered topically.
 22. Themethod of claim 1, wherein the agent is administered by infusion intothe kidneys.
 23. The method of claim 1, wherein the agent isadministered by infusion into the bladder.
 24. The method of claim 1,wherein the agent is administered by catheter into the bladder.
 25. Themethod of claim 1, wherein the administration of the agent havingsialidase activity causes one or more of: a reduction of dysuria, areduction of frequency of urination, a reduction of subrapublic pain, areduction of hematuria, a decrease in symptoms associated withnephropathy, and a reduction of BKPyV viral load.